Curcumin Assists Anti-EV71 Activity of IFN-α by Inhibiting IFNAR1 Reduction in SH-SY5Y Cells

Background and aim Enterovirus 71(EV71) can cause severe hand, foot, and mouth disease (HFMD) with brain tissue involvement. Few effective anti-EV71 drugs are presently available in clinical practice. Interferon-α (IFN-α) was ineffective while Curcumin was effective in restricting EV71 replication in non-neuronal cells. Ubiquitin-proteasome-mediated degradation of interferon-alpha receptor 1 (IFNAR1) protein contributes to IFN-α resistance. Current study aimed to determine synergistic inhibition of EV71 by Curcumin and IFN-α in human neuroblastoma SH-SY5Y cells. Methods SH-SY5Y cells were infected with mock-/Curcumin-pre-incubated EV71 or transfected with plasmid containing interferon-stimulated response element (ISRE) or mRNA containing viral internal ribosomal entry site (IRES) following by post-treatment with Curcumin with or without IFN-α. Supernatant IFN-α/β was detected by ELISA. ISRE, IRSE, proteasome and deubiquitinating activity were measured by luciferase assay. EV71 RNA and viral protein or IFNAR1 were determined by qPCR and western blot, respectively. Results EV71 flailed to completely block IFN-α/β production but inhibited IFN-α signal. Curcumin only slightly inhibited EV71 proliferation without modulating virus attachment and internalization. However, Curcumin addition restored IFN-α-mediated ISRE activity thus significantly inhibiting EV71 replication. Furthermore, EV71 also reduced IFNAR1 protein with proteasome-dependence in SH-SY5Y cells, which can be reversed by Curcumin addition with the evidence that it lowered proteasome activity. Conclusion These data demonstrate that Curcumin assists anti-EV71 activity of IFN-α by inhibiting IFNAR1 reduction via ubiquitin-proteasome disruption in SH-SY5Y cells

Translocating lipopolysaccharide correlates with the severity of enterovirus A71-induced HFMD by promoting pro-inflammation and viral IRES activity

Background The increase of inflammation-inducing enterobacteria was recently observed in severe hand, foot, and mouth disease (HFMD) caused by Enterovirus A71 (EV-A71). This study aimed to verify the occurrence of bacterial translocation (BT) and further explore the contributory role of BT to severity of EV-A71-mediated HFMD cases. Methods Serum specimens from 65 mild and 65 severe EV-A71-associated HFMD cases and 65 healthy children were collected. EV-A71 VP1 in serum, inflammatory mediators including C-reactive protein, IL-1β, IL-6, interferon-γ and tumor necrosis factor-α, BT related biomarkers including Claudin-3, intestinal fatty acid binding protein, lipopolysaccharide (LPS), soluble CD14 (sCD14) and endotoxin core antibody were measured by ELISA. Bacterial DNA (BactDNA) fragments were quantified by quantified PCR (qPCR). Rhabdomyosarcoma (RD) or SH-SY5Y cells, infected with LPS-pre-incubated EV-A71 or transfected with plasmid containing viral 2Apro or mRNA containing viral internal ribosomal entry site (IRES), were post-treated with or without LPS in vitro. EV-A71 RNA and viral or cellular proteins were determined by qPCR and western blot, respectively. Results Compared to mild HFMD patients, remarkably higher inflammatory mediators as well as BT-related biomarkers except BactDNA were observed in severe HFMD cases (all P < 0.05). In severe HFMD group, circulating concentrations of LPS and sCD14 showed statistical correlations with inflammation indices (all P < 0.05), serum levels of EV-A71 VP1 were found to be positively correlated with serum LPS (r = 0.341, P = 0.005) and serum sCD14 (r = 0.458, P < 0.001). In vitro, EV-A71 attachment and internalization were only slightly promoted by LPS pre-incubation; however, EV-A71 proliferation and viral 2Apro-mediated IRES activity were significantly accelerated by LPS post-treatment. Conclusions Our results collectively indicate that gut-derived translocating LPS contributes to the severity of EV-A71-induced HFMD by driving inflammatory response and viral proliferation via viral 2Apro-mediated IRES.

A Small Ligand That Selectively Binds to the G-quadruplex at the Human Vascular Endothelial Growth Factor Internal Ribosomal Entry Site and Represses the Translation

G-quadruplexes are believed to have important biological functions, so many small molecules have been screened or developed for targeting G-quadruplexes. However, it is still a major challenge to find molecules that recognize specific G-quadruplexes. Here, by using a combination of surface plasmon resonance, electrospray ionization mass spectrometry, circular dichroism, Western blot, luciferase assay, and reverse transcriptase stop assay, we observed a small molecule, namely, oxymatrine (OMT) that could selectively bind to the RNA G-quadruplex in 5′-untranslated regions (UTRs) of human vascular endothelial growth factor (hVEGF), but could not bind to other G-quadruplexes. OMT could selectively repress the translation of VEGF in cervical cancer cells. Furthermore, it could recognize VEGF RNA G-quadruplexes in special conformations. The results indicate that OMT may serve as a potentially special tool for studying the VEGF RNA G-quadruplex in cells and as a valuable scaffold for the design of ligands that recognize different G-quadruplexes.

Translocating lipopolysaccharide correlates with the severity of enterovirus A71-induced HFMD by promoting pro-inflammation and viral IRES activity

BackgroundThe increase of inflammation-inducing enterobacteria was recently observed in severe hand, foot, and mouth disease (HFMD) caused by Enterovirus A71 (EV-A71). This study aimed to verify the occurrence of bacterial translocation (BT) and further explore the contributory role of BT to severity of EV-A71-mediated HFMD cases. MethodsSerum specimens from 65 mild and 65 severe EV-A71-associated HFMD cases and 65 healthy children were collected. EV-A71 VP1 in serum, inflammatory mediators including C-reactive protein, IL-1β, IL-6, interferon-γ and tumor necrosis factor-α, BT related biomarkers including Claudin-3, intestinal fatty acid binding protein, lipopolysaccharide (LPS), soluble CD14(sCD14) and endotoxin core antibody were measured by ELISA. Bacterial DNA (BactDNA) fragments were quantified by quantified PCR (qPCR). Rhabdomyosarcoma (RD) cells, infected with LPS-pre-incubated EV-A71 or transfected with plasmid containing viral 2A pro or mRNA containing viral internal ribosomal entry site (IRES), were post-treated with or without LPS in vitro . EV-A71 RNA and viral or cellular proteins were determined by qPCR and western blot, respectively. ResultsCompared to mild HFMD patients, remarkably higher inflammatory mediators as well as BT-related biomarkers except BactDNA were observed in severe HFMD cases (all P <0.05). In severe HFMD group, circulating concentrations of LPS and sCD14 showed statistical correlations with inflammation indices (all P <0.05), serum levels of EV-A71 VP1 were found to be positively correlated with serum LPS ( r =0.341, P =0.005) and serum sCD14( r =0.458, P <0.001). In vitro , EV-A71 attachment and internalization were only slightly promoted by LPS pre-incubation; however, EV-A71 proliferation and viral 2A pro -mediated IRES activity were significantly accelerated by LPS post-treatment. ConclusionsOur results collectively indicate that gut-derived translocating LPS contributes to the severity of EV-A71-induced HFMD by driving inflammatory response and viral proliferation via viral 2A pro -mediated IRES.

A proposed division of the family Picornaviridae into subfamilies based on phylogenetic relationships and functional genomic organization

The highly diverse virus family Picornaviridae presently comprises 68 approved genera with 158 species plus many unassigned viruses. In order to better match picornavirus taxonomy to the functional and genomic groupings between genera, the establishment of five subfamilies (Caphthovirinae, Kodimesavirinae, Ensavirinae, Paavivirinae and Heptrevirinae) is proposed. The subfamilies are defined by phylogenetic analyses of 3CD (precursor of virus-encoded proteinase and polymerase) and P1 (capsid protein precursor) coding sequences and comprise between 7 and 22 currently approved virus genera. Due to the high within-subfamily and between-subfamily divergences of the picornavirus genera, p-distance estimates are unsuited for the demarcation of subfamilies. Members of the proposed subfamilies typically show some commonalities in their genome organisations, including VP1/2A cleavage mechanisms and possession of leader proteins. Other features, such as internal ribosomal entry site types, are more variable within and between members of genera. Some subfamilies are characterised by homology of proteins 1A, 2A, 2B and 3A encoded by members, which do not belong to the canon of orthologous picornavirus proteins. The proposed addition of a subfamily layer to the taxonomy of picornaviruses provides a valuable additional organisational level to the family that acknowledges the existence of higher-level evolutionary groupings of its component genera.

In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles

The 5′ untranslated region (5′ UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contains sequence homology to the HCV type III internal ribosome entry sites (IRES). Utilizing a monocistronic expression vector with an RNA polymerase I promoter to drive transcription, we show cell-specific IRES translation and regions within the IRES required for full functionality. Focusing on RHV, we further pseudotyped lentivirus with RHV and showed cell surface expression of the envelope proteins and transduction of murine hepatocytes and we then constructed full-length RHV and RPgV replicons with reporter genes. Using the replicon system, we show that the RHV NS3-4A protease cleaves a mitochondrial antiviral signaling protein reporter. However, liver-derived cells did not readily support the complete viral life cycle.

Analyses of Gnai3-iresGFP reporter mice reveal unknown Gαi3 expression sites

Inhibitory G proteins (Gi proteins) are highly homologous but play distinct biological roles. However, their isoform-specific detection remains challenging. To facilitate the analysis of Gαi3 expression, we generated a Gnai3- iresGFP reporter mouse line. An internal ribosomal entry site (IRES) was inserted behind the stop-codon of the Gnai3 gene to initiate simultaneous translation of the GFP cDNA together with Gαi3. The expression of GFP was confirmed in spleen and thymus tissue by immunoblot analysis. Importantly, the GFP knock-in (ki) did not alter Gαi3 expression levels in all organs tested including spleen and thymus compared to wild-type littermates. Flow cytometry of thymocytes, splenic and blood cell suspensions revealed significantly higher GFP fluorescence intensities in homozygous ki/ki animals compared to heterozygous mice (+/ki). Using cell-type specific surface markers GFP fluorescence was assigned to B cells, T cells, macrophages and granulocytes from both splenic and blood cells and additionally blood-derived platelets. Moreover, immunofluorescent staining of the inner ear from knock-in mice unraveled GFP expression in sensory and non-sensory cell types, with highest levels in Deiter’s cells and in the first row of Hensen’s cells in the organ of Corti, indicating a novel site for Gαi3 expression. In summary, the Gnai3- iresGFP reporter mouse represents an ideal tool for precise analyses of Gαi3 expression patterns and sites.

Enhanced virus translation enables miR-122-independent Hepatitis C Virus propagation

The 5’UTR of the Hepatitis C Virus genome forms RNA structures that regulate virus replication and translation. The region contains a viral internal ribosomal entry site and a 5’ terminal region. Binding of the liver specific miRNA, miR-122, to two conserved binding sites in the 5’ terminal region regulates viral replication, translation, and genome stability, and is essential for efficient virus replication, but its precise mechanism of its action is still under debate. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5’ UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable virus replication in cell culture, several viral variants with 5’ UTR mutations exhibit low level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Further, we also show that the mutant genomes display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Finally, we provide evidence that translation regulation is the major role for miR-122, and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5’ UTR mutations that stimulate translation, and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in the virus life cycle.

Revisiting the pathogenic mechanism of the GJB1 5’ UTR c.-103C > T mutation causing CMTX1

The second most common form of Charcot-Marie-Tooth neuropathy (CMT), X-linked CMT type X1 (CMTX1), is caused by coding and non-coding mutations in the gap junction beta 1 (GJB1) gene. The non-coding GJB1 c.-103C > T mutation (NM_000166.5) has been reported to cause CMTX1 in multiple families. This study assessed the internal ribosomal entry site (IRES) activity previously reported for the rat Gjb1 P2 5’ untranslated region (UTR). Using a bicistronic assay and transfecting RT4 Schwann cells, IRES activity of the human GJB1 P2 5’ UTR was compared to the GJB1 P2 5’ UTR containing either the c.-103C > T mutation or the non-pathogenic c.-102G > A variant. No differences in GJB1 P2 5’ UTR IRES activity were observed between the negative control, the wild-type P2 5’ UTR, the c.-103C > T 5’ UTR or the c.-102G > A 5’ UTR, irrespective of the GJB1 intron being present (p = .429 with intron, and p = .865 without). A theoretical c.-131A > G variant was predicted to result in the same RNA secondary structure as the GJB1 c.-103C > T P2 5’ UTR. However, no significant difference was observed between expression from the wild-type GJB1 P2 5’ UTR and the GJB1 c.-131A > G variant (p = .688). Deletion of the conserved region surrounding the c.-103C > T mutation (c.-108_-103del) resulted in significantly higher expression than the c.-103C > T mutation alone (p = .019), suggesting that the conserved c.-108_-103 region was not essential for translation. The reporter assays in this study do not recapitulate the previously reported GJB1 IRES activity and suggest an alternate pathogenic mechanism for the c.-103C > T CMTX1 non-coding mutation.